Screening of Combinatorial Libraries for Substrate Preference by

We present a rapid screening method for monitoring enzyme specificity using both combinatorial chemistry and mass spectrometry where, as an example, t...
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Anal. Chem. 2005, 77, 698-701

Screening of Combinatorial Libraries for Substrate Preference by Mass Spectrometry Stanley M. Stevens Jr.,†,§ Katalin Prokai-Tatrai,‡ and Laszlo Prokai*,†

Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida 32610-0485, and Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, Florida 32610-0267

We present a rapid screening method for monitoring enzyme specificity using both combinatorial chemistry and mass spectrometry where, as an example, the substrate specificity of peptidylglycine r-amidating enzyme was determined and compared against a conventional quantitative technique. Whereas alternative methods for library screening are generally limited to certain enzymes and can present difficulties in the synthesis or derivatization of potential substrates, the approach we call chirality-based isotope labeling for a library of substrates (CHILLS) does not fall short to such limitations, since we exploit the inherent stereospecificity of enzymes to determine preferred substrates. Additionally, the CHILLS method generates accurate results, as compared to typical screening procedures that require tedious method development, because the synthesized library contains a structurally similar internal standard for each individual library component in order to quantitate the progress of enzymatic reactions. Combinatorial chemistry has become a powerful tool for the discovery and optimization of ligands that bind to proteins, including receptors.1,2 Numerous high-throughput screening methods (phage display,3 quenched fluorescence,4 etc.) have been developed to determine optimal substrates of enzymes, yet these techniques are generally complicated and often limited to certain classes of enzymes and appropriate substrate probes.5,6 Rather than testing compounds individually, the use of combinatorial mixtures has been promising for the identification of optimal enzyme substrates.7-10 The syntheses of combinatorial mixtures have now been considered routine procedures.11 * To whom correspondence should be addressed. Phone: (352) 392-3421. Fax: (352) 392-9455. E-mail: [email protected]. † College of Pharmacy. ‡ College of Medicine. § Present address: Protein Chemistry and Molecular Biomarkers Core Facilities, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610-0156. (1) Ecker, D. J.; Crooke, S. T. Bio/Technology 1995, 13, 351-360. (2) Williard, X.; Pop, I.; Bourel, L.; Horvath, D.; Baudelle, R.; Melnyk, P.; Deprez, B.; Tarter, A. Eur. J. Med. Chem. 1996, 31, 87-98. (3) Smith, G. P.; Petrenko, V. A. Chem. Rev. 1997, 97, 391-410. (4) Westphal, V.; Spetzler, J. C.; Meldal, M.; Christensen, U.; Winther, J. R. J. Biol. Chem. 1998, 273, 24992-24999. (5) Labrijn, A. F.; Koppelman, M. H. G. M.; Verhagen, J.; Brouwer, M. C.; Schuitemaker, H.; Hack, C. E.; Huisman, H. G. J. Immunol. Methods 2002, 261, 37-48. (6) Hu, Y. G.; Wei, Y.; Zhou, Y.; Rajagopalan, P. T. R.; Pei, D. Biochemistry 1999, 38, 643-650.

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During a catalytic event, an enzyme binds to a substrate, converts the substrate into product(s), and then dissociates from the product(s). Measuring directly, however, the interaction that occurred between the enzyme and the substrate/product(s) is practically impossible. When an enzyme catalyzes the reactions of multiple substrates in the same solution, the relative activities toward the substrates are determined by the substrate concentrations and the ratios of the kcat/Km values for the substrates.12 Because mixture-based combinatorial approaches are usually based on equimolar or practically equimolar concentrations of the compounds in the library,11 conversion rates should reflect activities according to the kcat/Km values. A major challenge exists in separation and rapid identification of individual compounds from complex (combinatorial) mixtures. Mass spectrometry (MS) has been the method of choice for the rapid identification and quantification of the substrates and their corresponding reaction products. On the other hand, tedious method development to obtain accurate data has hampered the widespread exploitation of MS-based screening techniques for substrate specificity of enzymes. Here we report a method employing a combination of library design and the use of reversed-phase high-performance liquid chromatography (LC) coupled with electrospray ionization (ESI) MS to address shortcomings of the mixture-based approach to reliably assess substrate preference in enzyme-catalyzed reactions. As a model, we investigated peptidylglycine R-amidating monooxygenase (PAM) involved in the C-terminal amidation of glycine-extended prohormones.13 EXPERIMENTAL DETAILS The screening mixture library Ac-Arg-Gln-Leu-(Xaa)-Gly-OH, where Xaa represents either Phe, Tyr, Trp, Ile, Leu, Val, or Pro in position 4 of the peptide, was prepared by solid-phase peptide synthesis (SPPS) using 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry and the split-and-mix method.14 (The three-letter ab(7) Wigger, M.; Nawrocki, J. P.; Watson, C. H.; Eyler, J. R.; Benner, S. A. Rapid Commun. Mass Spectrom. 1997, 11, 1749-1752. (8) Hinderling, C.; Chen, P. Angew. Chem., Int. Ed. 1999, 38, 2253-2256. (9) Walk, T. B.; Trautwein, A. W.; Richter, H.; Jung, G. Angew. Chem., Int. Ed. 1999, 38, 1763-1765. (10) Wang, P. G.; Snavley, D. F.; Freitas, M. A.; Pei, D. Rapid Commun. Mass Spectrom. 2001, 15, 1166-1171. (11) Houghten, R. A.; Pinilla, C.; Appel, J. R.; Blondelle, S. F.; Dooley, C. T.; Eichler, J.; Nefzi, A.; Ostresh, J. M. J. Med. Chem. 1999, 42, 3743-3778. (12) Abeles, R. H.; Frey, P. A.; Jenck, W. P. Biochemistry; Jones and Bartlett: Boston, 1992; p 84. (13) Perkins, S. N.; Husten, E. J.; Eipper, B. A. Biochem. Biophys. Res. Commun. 1990, 171, 926-932. 10.1021/ac0489925 CCC: $30.25

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breviations were used for identifying the amino acid residues.) All reagents were of synthesis grade and available commercially. Briefly, Fmoc-Xaa-OH (with appropriate orthogonal side-chain protection) was coupled to 0.1 g of preloaded Gly-Wang resin using benzotriazole-1-yl-oxy-tris-pyrrolidinophosphoniumhexafluorophosphate/N-hydroxybenztriazole/diisopropylethylamine (PyBOP/HOBt/DIPEA, 1:1:2 molar ratio) activation for 4 h. After repeating the coupling, the resins were washed with dimethylformamide (DMF, 3×), methanol (3×), and DMF again (3×) and were combined. The Fmoc protecting group was removed by treating the resin with piperidine (20% v/v) in DMF for 10 min, and then the peptide chain was elongated using standard Fmocbased SPPS. Double coupling was always used to ensure completion of the reaction. In the final step, the peptide chain was terminated by acetylation with acetic anhydride in pyridine for 20 min. The library was removed from the resin using trifluoroacetic acid/water, precipitated, and washed with diethyl ether. The solidified precipitate was dissolved in water and extracted with ethyl acetate, and the aqueous phase was freeze-dried. ESI-MS characterization and correlation with simulated mass distribution15 confirmed the presence of practically equimolar concentrations of the expected peptides in the combinatorial mixture. A chiralitybased isotope labeling for a library of substrates (CHILLS) approach was performed by synthesizing a mixture of selectively labeled (d3-Ac)-Arg-Gln-Leu-(Xaa/D-Xaa)-Gly-OH by the splitand-mix method, in which equivalent amounts of the resin-bound Ac-Arg-Gln-Leu-(Xaa)-Gly-OH and (d3-Ac)-Arg-Gln-Leu-(D-Xaa)Gly-OH were mixed only before cleaving the peptide mixture from the resin. (Acetic anhydride-d6 was obtained from Aldrich, Milwaukee, WI.) A small portion of the resin containing the deuterioacetyl-terminated D-Xaa peptide mixture was cleaved separately to obtain (d3-Ac)-Arg-Gln-Leu-(D-Xaa)-Gly-OH as a control library. Ac-Arg-Gln-Leu-Gly-OH and Ac-Arg-Gln-IleGly-OH was also prepared individually for the unambiguous identification of these isomers. The enzyme assay16 was carried out at 37 °C by adding 450 U of the enzyme [PAM, 18 µL from the 25 000 U/mL stock solution purchased from Unigene (Fairfield, NJ)] into the preincubated solution (1 mL) of 100 mM MES/KOH, pH 6.0, 1.5 mM sodium ascorbate, 0.001% (v/v) Triton X-100, 30 mM KCl, 30 mM KI, 1 µM CuSO4, 100 µg/mL catalase and 1 mM of the combinatorial libraries. Aliquots were taken at regular time periods and added to 100 µL of ice-cold aqueous 10% (v/v) acetic acid to stop the reaction. The sample was then desalted (C18 ZipTip), lyophilized, and reconstituted in 0.5% acetic acid prior to LC/MS analysis. When samples were removed from the incubation with the screening of the control [(d3-Ac)-Arg-Gln-Leu-(D-Xaa)-Gly-OH] library, the stopping solution contained 20 µM internal standard (Trigonellyl-Tyr-D-Ala-Gly-Phe-D-Leu-OH, prepared by standard, Fmoc-based SPPS17); however, no internal standard was used for the samples removed from the mixture containing the CHILLS. (14) Furka, A.; Sebestyen, F.; Asgedom, M.; Dibo, G. Int. J. Pept. Protein Res. 1991, 37, 487-493. (15) Steinbeck, C.; Berlin, K.; Richert, C. J. Chem. Inf. Comput. Sci. 1997, 37, 449-457. (16) Kulathila, R.; Consalvo, A. P.; Fitzpatrick, P. F.; Freeman, J. C.; Snyder, L. M.; Villafranca, J. J.; Merkler, D. J. Arch. Biochem. Biophys. 1994, 311, 191-195. (17) Prokai-Tatrai, K.; Prokai, L.; Bodor, N. J. Med. Chem. 1996, 39, 47754782.

Microbore reversed-phase HPLC separation of the peptide libraries was performed on a 15 cm × 0.5 mm i.d. Targa C18 (Higgins Analytical, Mountain View, CA) column. A sample loop size of 5 µL was used in combination with a MicroPro gradient solvent delivery system (Eldex Laboratories, Napa, CA) that provided gradient flow rates at 10 µL/min to the mass spectrometer. Solvent A was 0.5% acetic acid, 5% acetonitrile, and 95% water, whereas solvent B was 0.5% acetic acid, 5% water, and 95% acetonitrile. After column equilibration in 100% solvent A, the sample was injected. Following a 3-min isocratic solvent delivery with 100% solvent A, a linear gradient was carried out for 30 min to 40% solvent B. ESI experiments were performed on a quadrupole ion trap (IT) instrument (LCQ, ThermoFinnigan, San Jose, CA) operated with the Xcalibur (version 1.3) data system software. ESI spray voltage and capillary temperature were maintained at 4.5 kV and 200 °C, respectively, using a sheath gas flow of 60 arbitrary units to aid in desolvation. Full-scan mass spectra were acquired from m/z 500 to 750 using the automatic gain control mode of ion trapping (target ion count of 5 × 107). To verify peptide structure, product ion mass spectra were obtained by collision-induced dissociation (CID) (1.5 u isolation width, 1.75 V activation amplitude) employing helium as the target gas. RESULTS AND DISCUSSION Regulation of biological function at the protein level can be controlled by numerous posttranslational modifications.18 Of the many posttranslational modifications reported and characterized, the most imperative one that initiates biological activity of approximately one-half of the known peptide hormones is Cterminal amidation.19-21 Conversion of glycine-extended prohormones to their biologically active analogues is accomplished by the peptidylglycine R-amidating monooxygenase (PAM) enzyme.22,23 The PAM gene actually encodes two product enzymes, peptidylglycine R-hydroxylating monooxygenase (PHM) and peptidyl-R-hydroxyglycine R-amidating lyase (PAL), both of which are involved in the two-step reaction mechanism of PAM shown in Scheme 1. The first step in this mechanism involves hydroxylation of the R-pro-S hydrogen of a glycine-extended propeptide by PHM. A second enzyme PAL is then required for lysis of the R-hydroxyglycine analogue to form an activated R-carboxyamidopeptide and glyoxylate. In the control screening experiments, a model combinatorial library of Ac-Arg-Gln-Leu-(Xaa)-Gly-OH (Xaa ) Ile, Leu, Phe, Pro, Tyr, Trp, or Val) and a single internal standard-based monitoring of the decrease in substrate concentrations were used. The peptide sequence was derived from Gln-His-Pro-Gly, a progenitor sequence for thyrotropin-releasing hormone (TRH) in the prepro-TRH,24 and used successfully by us for creating CNSpermeable prodrugs for a TRH analogue.25 The library was limited to hydrophobic amino acids preceding Gly, since our preliminary (18) Nakai, K. J. Struct. Biol. 2001, 134, 103-116. (19) Eipper, B. A.; Stoffers, D. S.; Mains, R. E. Annu. Rev. Neurosci. 1992, 15, 57-85. (20) Merkler, D. J. Enzyme Microbiol. Technol. 1994, 16, 450-456. (21) Fan, X. M.; Spijker, S.; Akalal, D. B. G.; Nagle, G. T. Mol. Brain Res. 2000, 82, 25-34. (22) Perkins, S. N.; Husten, E. J.; Eipper, B. A. Biophys. Res. Commun. 1990, 171, 926-932. (23) Martı´nez, A.; Treston, A. M. Mol. Cell. Endocrinol. 1996, 123, 113-117. (24) National Center for Biotechnology Information (NCBI) protein database accession number: 207468. NCBI website available at http:// www.ncbi.nlm.nih.gov.

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Scheme 1. Mechanism of Carboxy-Terminal Amidation of Gly-Extended Peptides by PAM

experiments indicated the preference of PAM for the amidation of these specific residues (data not shown). Figure 1 illustrates that, given the presence of isomers/isobars in the library including Xaa ) Leu/Ile and possible overlaps of monoisotopic masses with 13C and 15N peaks of other components, LC coupled with ESI/ MS was needed to separate those components having the same nominal mass. The area under the trace was then determined and compared to the trace area of a single “spiked” internal standard in order to quantify the loss of substrate over time. However, we noticed huge variation in the kinetic curves due to the use of a single internal standard compound upon LC/ESI-MS analysis, which made a reliable “ranking” of substrates nearly impossible. In contrast, subtle differences in the conversion rates could be detected by using the method described below. We extended the initial library of Ac-Arg-Gln-Leu-(Xaa)-GlyOH with (d3-Ac)-Arg-Gln-Leu-(D-Xaa)-Gly-OH (where Xaa represented Ile, Leu, Phe, Pro, Tyr, Trp, or Val) to utilize PAM stereospecificty26 for an improved screening approach. This new combinatorial mixture of Gly-extended model peptides included penultimate L-amino acid residues as well as the counterpart peptides containing interchangeable D-amino acid residues that

Figure 1. (a) Reconstructed ion chromatograms for the combinatorial mixture Ac-Arg-Gln-Leu-Xaa-Gly-OH (Xaa ) Ile, Leu, Phe, Pro, Tyr, Trp, or Val), as well as the internal standard. TrigonellylTyr-D-Ala-Gly-Phe-D-Leu-OH. Baseline separation is demonstrated for the two isomers Ac-Arg-Gln-Leu-Ile-Gly-OH and Ac-Arg-GlnLeu-Leu-Gly-OH at m/z 628.3. 700 Analytical Chemistry, Vol. 77, No. 2, January 15, 2005

are normally not amidated by PAM.26 Since PAM exhibits no affinity for the peptides containing the unnatural amino acid residue (as confirmed by a separate experiment), the D-analogues remain unchanged throughout the incubation of the mixture with the enzyme. Therefore, the latter can be used as an internal reference to quantify the progress of the PAM enzymatic reaction by LC/MS. Enantiomers of substrates may also be enzyme inhibitors,27 but their presence should not interfere with the ranking of substrate-library components in mixture-based experiments once appropriate experimental conditions, such as low substrate/inhibitor concentrations, are chosen to avoid a significant degree of enzyme inhibition. This method alleviates tedious and time-consuming LC/MS assay development, which otherwise would include finding internal standard(s) structurally similar to all components in the library and performing assay calibrations. This method is simplified by the determination of ion intensity or chromatographic peak area ratios. A similar practice has been widely used in today’s proteomics analyses that employ, for example, isotope-coded affinity tags28 or proteolytic stable-isotope labeling29 for differential quantification. Although accurate mass and high mass resolving power can be beneficial for the analysis of combinatorial and peptide libraries,30,31 distinction between D- and L-isomers cannot be accomplished without chromatographic separation prior to mass spectrometric detection. Unfortunately, the need for adequate chromatographic separation can sometimes decrease the throughput of the LC/MS method. However, if chromatographic separation is not possible or a rapid-gradient approach is favored to increase throughput, isotopic labeling of selected library components may be employed. (The labeling method should be chosen (25) Prokai, L.; Prokai-Tatrai, K.; Ouyang, X.; Kim, H.-S.; Wu, W.-M.; Zharikova, A.; Bodor, N. J. Med. Chem. 1999, 42, 4563-4571. (26) Ping, D.; Mounier, C. E.; May, S. W. J. Biol. Chem. 1995, 270, 2925029255. (27) Ingles, D. W.; Knowles, J. R. Biochem. J. 1967, 104, 369-377. (28) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (29) Yao, X. D.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-2842. (30) Ramjit, H. G.; Kruppa, G. H.; Speir, J. P.; Ross, C. W.; Garsky, V. M. Rapid Comm. Mass Spectrom. 2000, 14, 1368-1376. (31) Gao, J. M.; Cheng, X. H.; Chen, R. D.; Sigal, G. B.; Bruce, J. E.; Schwartz, B. L.; Hofstadler, S. A.; Anderson, G. A.; Smith, R. D.; Whitesides, G. M. J. Med. Chem. 1996, 39, 1949-1955.

Table 1. PAM Enzymatic Rate Constants (( Standard Deviation) Determined for Peptides Present in the Ac-Arg-Gln-Leu-(Xaa)-Gly-OH Library by the CHILLS Method and LC/ESI-MS. Xaa

k (min-1)

preference ratio (k/kref)a

Phe Trp Tyr Ile Val Leu

0.090 ( 0.007 0.070 ( 0.015 0.063 ( 0.005 0.017 ( 0.005 0.012 ( 0.002 0.012 ( 0.004

1.00 0.77 0.70 0.19 0.13 0.13

a k ref represents the highest rate constant value (calculated for Xaa ) Phe).

incubation (up to 60 min). Figure 2b and 2c displays the plot of substrate concentration relative to the D-analogue versus incubation time with PAM. As illustrated in these graphs, reasonable curves can be generated, when compared to the conventional internal standard method, to monitor progress of the enzymatic reaction and ultimately determine the best substrate from the library, essentially without prior LC/MS method development by measuring substrate to internal standard ion abundance ratios. In addition to improved reaction rate data, simultaneous determination of enzyme specificity for multiple substrates in a mixture can be accomplished with high confidence by this method. Data obtained by our analyses indicated that the optimal substrate from the library was Ac-Arg-Gln-Leu-Phe-Gly-OH, which implicated that PAM favored penultimate aromatic residues (specifically Phe, followed by Trp and Tyr) over nonpolar residues. Similar findings for substrate preference have been reported by others on the basis of the inhibitory potency of tripeptide substrates measured individually in competitive kinetic assays.32 Table 1 lists the rate constants calculated for each individual peptide present in the library. Ac-Arg-Gln-Leu-Pro-Gly-OH was essentially unreactive toward PAM in the combinatorial mixture tested. Figure 2. (a) Reconstructed ion chromatograms illustrating the separation of a substrate (Ac-Arg-Gln-Leu-Phe-Gly-OH), its internal reference (d3-Ac)-Arg-Gln-Leu-D-Phe-OH, and the enzymatic reaction product Ac-Arg-Gln-Leu-Phe-NH2 following incubation of a chirality-based isotope-labeled library of substrates (CHILLS) with PAM for 18 min. Substrate concentration profile versus time using the conventional single internal standard method and the D-analogue of the substrate as the internal standard are shown in (b) and (c), respectively.

appropriately to avoid the introduction of isotope effect.) In our PAM screening library, we acylated the N terminus of the D-isomers by using deuterium-labeled acetic anhydride (an inexpensive, commercially available reagent). Figure 2a shows the selected ion retrieval chromatograms for Ac-Arg-Gln-Leu-PheGly-OH (substrate), (d3-Ac)-Arg-Gln-Leu-D-Phe-Gly-OH (internal standard), and the reaction product Ac-Arg-Gln-Leu-PheNH2 upon incubation of the combinatorial mixture with PAM for 18 min. Again, there was no evidence from the assay for the formation of any product from the D-analogue (with expected molecular ion at m/z 607.3) throughout the time course of the (32) Shimo, H.; Kawahara, T.; Suzuki, K.; Iwasaki, Y.; Jeng, A. Y.; Nishikawa, Y. Eur. J. Biochem. 1992, 209, 189-194.

CONCLUSION The methodology of chirality-based isotope labeling combined with the use of routine LC/MS as the assay method to rapidly find preferred substrates has been demonstrated. CHILLS can conceivably be expanded to many enzymes for which substrate chirality is an important factor in the catalytic mechanism. The performance of today’s LC/MS instruments should allow for testing libraries containing hundreds of enzyme substrates simultaneously by this method. The additional effort needed to employ the CHILLS method, when compared to the straightforward splitand-mix or reagent-mixture procedure, has a much bigger return in saving time and expensive resources, because the technique apparently minimizes the time necessary for method development and reduces the error associated with conventional assay protocols based on LC/MS. ACKNOWLEDGMENT This work was supported by the National Institutes of Health (MH59360). Received for review July 10, 2004. Accepted October 19, 2004. AC0489925 Analytical Chemistry, Vol. 77, No. 2, January 15, 2005

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